Approximately two million deaths a year result from infections with the malaria parasite Plasmodium falciparum, the most deadly human parasitic disease. Drug-resistant strains of this parasite have emerged and threaten the utility of conventional anti-malarial compounds used to fight this disease. In a variety of fungal species, it has been shown that Hsp90 inhibitors can reverse drug resistance, and this may be true for other eukaryotic pathogens such as P. falciparum. Furthermore, Hsp90 inhibitors show potent anti-malarial activity against Plasmodium strains in culture. As global inhibition of Hsp90 may be harmful in disease-compromised individuals, here we propose a strategy to discover compounds that specifically inhibit the malarial homolog of this protein. We plan to execute a high throughput screen for compounds that possess the desired selectivity by using yeast strains that have been genetically engineered to survive on either the P. falciparum (Pf) or human (Hs) Hsp90 homolog that replaces the native fungal protein. Compounds that significantly inhibit the growth of PfHsp90 yeast will be counter-screened to find those that do not significantly inhibit growth of the HsHsp90 strain. Preliminary results against known Hsp90 inhibitors as well as a test set of compounds show that this is a very robust, repeatable, and cost efficient assay strategy. Additionally, hits from the test set show the desired species specificity and provide excellent proof of concept. Secondary assays will include testing hit compounds against P. falciparum-infected erythrocytes to define the anti-parasite activity of these compounds, as well as standard viability assays to determine cytotoxicity in human cell lines. The effect of these compounds on Hsp90 will be probed through yeast-based assays to assess the function of proteins known to require Hsp90 to fold. Up to ten hits will be selected for further biological study, and a library of related analogs will be assembled to define structure activity relationships. Compounds discovered through this screening strategy will meet a critical need for probes to dissect the contribution of parasite-encoded Hsp90 from that of the host red blood cell in the complex life cycle of P. falciparum. In future work they could well serve as valuable leads for the development of anti-malarial drugs with a previously unexploited mode of action. PUBLIC HEALTH RELEVANCE: The goal of this project is to identify inhibitors of the protein Hsp90 specific to the malarial species Plasmodium falciparum. As strains of P. falciparum have developed resistance to conventional anti-malarial drugs and spread worldwide, creating drugs that operate through a novel mechanism is critically important. Previous work shows both that inhibition of Hsp90 in fungi can reverse drug resistance and that inhibitors of Hsp90 are very potent in killing P. falciparum in infected red blood cells, therefore new drugs that target Hsp90 may provide an excellent method to combat this deadly disease.